Seminiferous Cord Formation and Germ-Cell Programming Epigenetic Transgenerational Actions of Endocrine Disruptors MICHAEL K. SKINNER AND MATTHEW D. ANWAY Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4231, USA ABSTRACT: The molecular and cellular control of embryonic testis develop- ment was investigated through an analysis of the embryonic testis transcrip- tome to identify potential regulatory factors for male sex determination and testis morphogenesis. One critical factor identified is neurotropin 3 (NT3). At the onset of male sex determination, Sertoli cells initiate differentiation and express NT3 to act as a chemotactic factor for mesonephros cells to migrate and associate with Sertoli-germ cell aggregates to promote cord formation. Promot- er analysis suggests that NT3 may be an initial downstream gene to SRY and helps promote testis morphogenesis. Endocrine disruptors were used to poten- tially interfere with embryonic testis development and further investigate this biological process. The estrogenic pesticide methoxychlor and antiandrogenic fungicide vinclozolin were used. Previous studies have shown that methoxy- chlor and vinclozolin both interfere with embryonic testis cord formation and cause increased spermatogenic cell apoptosis in the adult testis. Interestingly, transient in vivo exposure to endocrine disruptors at the time of male sex determination caused a transgenerational phenotype (F1-F4) of spermatogenic cell apoptosis and subfertility. This apparent epigenetic mechanism involves altered DNA methylation and permanent re-programming of the male germ- line. A series of genes with altered DNA methylation and imprinting are being identified. Observations reviewed demonstrate that a transient embryonic in utero exposure to an endocrine disruptor influences the embryonic testis tran- scriptome and through epigenetic effects (e.g., DNA methylation) results in abnormal germ-cell differentiation that subsequently influences adult sper- matogenic capacity and male fertility, and that this phenotype is transgenera- tional through the germ-line. The novel observations of transgenerational epigenetic endocrine disruptor actions on male reproduction critically impact the potential hazards of these compounds as environmental toxins. The litera- ture reviewed provides insight into the molecular and cellular control of embryonic testis development, male sex determination, and the programming of the male germ-line. Address for correspondence: Michael K. Skinner, Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4231. Voice: 509-335- 1524; fax: 509-335-2176. [email protected] Ann. N.Y. Acad. Sci. 1061: 18–32 (2005). © 2005 New York Academy of Sciences. doi: 10.1196/annals.1336.004 18 SKINNER & ANWAY: ACTIONS OF ENDOCRINE DISRUPTORS 19 KEYWORDS: endocrine disruptors; vinclozolin; methoxychlor; testis; sertoli; spermatogenesis; male fertility; epigenetic; transgenerational; sex determina- tion; review INTRODUCTION Transgenerational Epigenetic Phenomena Transgenerational effects of irradiation, chemical treatments (e.g., chemo- therapy), and environmental toxins such as endocrine disruptors have been observed over the last decade. Most transgenerational observations are simply the effects of the agent on the gestating mother and subsequent actions on the offspring associated with the F1 generation.1–3 Transgenerational effects for multiple generations have not been as thoroughly studied and will require transmission through the germ-line. In the context of the current review, transgenerational denotes germ-line transmis- sion to multiple generations, minimally the F2 generation. The ability of an external agent to induce a transgenerational effect is through an epigenetic phenomenon involving DNA methylation or stable chromosomal alterations.4–6 Transgeneration- al effects of irradiation were the first to be identified, and some have been shown to be transmitted through the germ-line to multiple generations.4,5,7 These are often associated with mutagenesis and tumor formations in subsequent generations. The treatment of cancer with chemotherapeutics also has been shown to cause transgen- erational effects,8–10 but the impact on multiple generations has not been thoroughly investigated. Recently, nutritional effects on the F1 generation have been ob- served.11 Environmental toxins such as endocrine disruptors have also been shown to influence the F1 generation after parental exposure,9,12–14 but studies have not demonstrated transgenerational effects on multiple generations. However, the poten- tial impact of such transgenerational effects of endocrine disruptors has been discussed.15 Epigenetic alterations that lead to transgenerational transmission of specific genetic traits or molecular events (e.g., imprinting) were recently identified.16–19 These observations have led to the conclusion that re-programming through an altered methylation state of the germ-line is responsible. The impact this has on human health and evolutionary importance is significant.16,17 Recent investigations of the DNA methylation state of primordial germ cells (PGCs) have indicated that as PGCs migrate down the genital ridge, de-methylation (i.e., erasure of methylation) begins and upon colonization in the early gonad complete de-methylation is achieved.20–22 This has primarily been observed through analysis of specific im- printed genes.23 During the period of sex determination in the gonad, germ cells un- dergo re-methylation involving sex-specific determination of the germ cells. Although de-methylation may not require the gonad somatic cells,21 re-methylation of the germ-line appears to be dependent on association with the somatic cells in the gonads.20,22 Due to this unique property of germ cells to undergo de-methylation and re-methylation during the period of sex determination in the developing gonad, the ability of an environmental agent such as an endocrine disruptor to influence through an epigenetic process the germ-line is postulated. This epigenetic effect on the germ-line could re-program the germ cell through an event such as altered DNA 20 ANNALS NEW YORK ACADEMY OF SCIENCES imprinting. This epigenetic effect could cause a transgenerational effect on subse- quent generations through the germ-line. Because re-methylation of the germ-line appears to depend upon gonadal somatic cells, an alteration in somatic cell function by an agent such as an endocrine disruptor could indirectly influence germ-cell re-methylation. Recent observations identify for the first time a transgenerational effect of endo- crine disruptors on testis development and adult spermatogenic cells.24 The endo- crine disruptors vinclozolin and methoxychlor have been shown to alter testis morphogenesis at the same time that germ-cell re-methylation occurs. Transient embryonic exposure of the endocrine disruptors at the time of gonadal sex determi- nation promoted an adult testis phenotype of increased spermatogenic cell apoptosis and decreased sperm concentration, and this phenotype was transmitted out to the fourth generation (F1-F4).24 This transgenerational phenotype was apparently due to altered DNA methylation of a number of genes identified.24 Reproductive Toxicology and Endocrine Disruptors Many reports have suggested that environmental endocrine disruptors, which act to mimic estrogens or act as antiestrogens or antiandrogens, are detrimental to repro- duction and may promote abnormalities such as a decrease in sperm count, an in- crease in testicular cancer,25,26 and an increase in abnormalities in sex determination in many species.27 Examples of the environmental endocrine disruptors that have been targeted for adverse effects on the reproductive systems in humans and animals are pesticides (e.g., methoxychlor), fungicides (e.g., vinclozolin), a range of xenoestrogens, and certain phthalates. Most of these chemicals are ubiquitous in the environment, and both humans and animals are exposed to them daily. Many of these compounds and endocrine disruptors can be metabolized into both estrogenic and antiandrogenic activities.28 Recently, methoxychlor and vinclozolin were used29–31 as model endocrine disruptors32 that have both estrogenic and antiandrogenic metabolites.28 Many environmental endocrine disruptors are weakly estrogenic and elicit their actions through estrogen receptors. The two mammalian receptors for estrogen (ER- α and ER-β) are widely distributed throughout the reproductive tract and during fetal gonad development.33,34 ER-β is present in higher concentrations within the fetal testis and ovary, whereas ER-α is present mainly within the uterus.35,36 During fetal testis development ER-β is first expressed in Sertoli and myoid cells after seminif- erous cord formation.37 In rats, ER-β has also been localized to pre-spermatogonia, which may explain the proliferative actions of estrogen on early postnatal gonocyte cultures.38 The importance of ER-α was further delineated when knockout mice39 and human males40 lacking expression of this gene were found to be sterile. Fetal development of the testis in these experiments was not altered. Early embryonic testis morphology in a double knockout remains to be examined.41 Neonatal expo- sure to estrogen alters ER-α and ER-β expression during postnatal testis and hypothalamic/pituitary development.42,43 Interestingly, neonatal exposure to the estrogenic compound